The main focus of this project is to determine the relative contributions of the thymus and the intestine in determining the intestinal intraepithelial T lymphocyte (IEL) repertoire. We have shown that IEL can be thymus-derived and we will use this system to test the hypothesis that T cell receptor (TCR)alpha-beta IEL are selected outside of the thymus. Our preliminary results suggest that extrathymic expression of class l major histocompatibility complex (MHG) proteins is required for positive selection of TCRalpha-beta IEL. However, we do not know where and when initial TCR gene rearrangement and lEL selection occur. Thymus grafting experiments using MHC-deficient hosts and TCR transgenic mice will answer the question of whether IEL are selected in the thymus prior to trafficking to the intestine. In situ analysis in model systems will determine whether extrathymic TCR rearrangement can occur. We will also examine the role of normal flora in determining the TCRalpha-beta IEL oligoclonal repertoire. The TCR repertoire of TCRalpha-beta IEL from gnotobiotic mice will be subjected to molecular analysis to determine the influence of flora on generation of TCR junctional diversity. Finally, we will produce TCR transgenic mice expressing IEL-type gamma-delta and alpha-beta TCRs to further study selection and development of IEL. Overall, this proposal presents a comprehensive and critical analysis of thymic versus extrathymic IEL selection and the factors involved in these processes.